癌症生物学

Cell viability and cytotoxicity assays are commonly used to investigate protein function and to evaluate drug efficacy in cancer and other disease models. Cytotoxicity is the measure of dead or damaged cells and is often quantified using assays based on cellular characteristics such as membrane integrity or mitochondrial metabolism. However, these assays are typically limited to endpoint analysis and lack emulation of physiological conditions. The IncuCyte Live and Dead Cell assay described here leverages common cell permeability methodologies but uses fluorescence microscopy channels to image both live and dead cells over time and phase microscopy channels to measure confluency. Cytotox green reagent is a cell membrane–impermeable dye that can only be taken up by cells with poor cell membrane integrity. NucLight rapid red dye is a cell membrane–permeable nuclear dye that can be taken up by all cells. Based on dye uptake and fluorescence intensity, the IncuCyte software can be used to analyze images for live and dead cell detection and quantification. Phase microscopy is used to determine confluency and can be further quantified using the IncuCyte software. We provide an application of this assay, using it to calculate IC₅₀ and EC₅₀ values for the assessment of drug efficacy.

Silver nanoparticles have been widely studied to possess antimicrobial as well as anticancer activity, and have found its applications in various fields including pharmaceutical industry, diagnostics, drug delivery, food industry, and others. For this purpose, several cell proliferation assays are widely used for the evaluation of anticancer activity of synthetic compounds as well as natural plant extracts. In general, a compound is said to possess an anticancer activity if it prevents the cancer cells to grow and divide actively, and indirectly activates the generic program of cell death. In this protocol, Alamar blue and MTT assay are described for the analysis of metabolic function and health of the cell. These procedures are generally used for the endpoint analysis. A549 cells are seeded in a 96-well plate, and after the adherence of the cells, they are treated with different concentrations of silver nanoparticles. Followed by 24 h of incubation, colorimetric dyes are added to the wells, and the absorbance is recorded to quantify the percentage cytotoxicity in the sample wells.

In this protocol, we describe a method to monitor cell proliferation and death by live-cell imaging of propidium iodide (PI)-stained adherent mammalian cells. PI is widely used to assess cell death. However, it is usually used in end-point assays. Recently, we implemented the use of PI for real-time cell death assessment by automated imaging. Cells are seeded in a 96-well format, and after attachment, the treatments are added directly to the wells together with PI. Thereafter, cells are subjected to automated time-lapse imaging and quantification by computer software. Combined analyses of phase-contrast and fluorescence images allow assessment of treatment effects on cell proliferation as well as the extent and kinetics of cell death.

This protocol is a flow cytometry-based method to measure the phagocytosis efficiency of necroptotic target cells by bone marrow-derived dendritic cells (BMDCs) in vitro (Aaes et al., 2016). The method is a slightly modified and updated version of the protocols used in previously published papers (Krysko et al., 2006; Brouckaert et al., 2004). In brief, the target cells are labeled with a CellTracker^TM dye before they are induced to undergo cell death. After a co-culture period of 2 h with BMDCs, the cells are immunostained with a dendritic cell marker and dead cell marker, and the phagocytic efficiency is quantified using a flow cytometer. This protocol can readily be used for target cells undergoing cell death modalities other than necroptosis.

This protocol utilizes PicoGreen 96-well plate technology. This method is applied to estimate the sensitivity of different tumor cell lines to chemodrugs.

Clonogenic assays serve as a useful tool to test whether a given cancer therapy can reduce the clonogenic survival of tumor cells. A colony is defined as a cluster of at least 50 cells that can often only be determined microscopically. A clonogenic assay is the method of choice to determine cell reproductive death after treatment with ionizing radiation, but can also be used to determine the effectiveness of other cytotoxic agents. The following protocol has been modified from a published version (Franken et al., 2006).